DNA encoding human follicle stimulating hormone receptor, vectors and cells containing such DNA, and methods of use thereof

ABSTRACT

The present invent embraces essentially pure human FSH receptor, or a fragment or mutant thereof which binds FSH, DNA encoding said receptor, fragment or mutant, expression vectors comprising said DNA, cells transfected with said expression vectors, and methods of producing said receptor, fragment or mutant by culturing said transfected cells. The present invention also includes pharmaceutical compositions comprising said receptor, fragment or mutant, as well as methods of treating patients with such compositions to reduce endogenous FSH bioactivity. An improved assay for human FSH using the receptor, fragment or mutant of the present invention is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.07/670,085, filed Mar. 15, 1991.

BACKGROUND OF THE INVENTION

This invention relates to human follicle stimulating hormone receptorand its synthesis by recombinant DNA techniques.

Follicle stimulating hormone (FSH) is a pituitary-derived heterodimericglycoprotein hormone which shares structural similarities withluteinizing hormone (LH) and thyroid stimulating hormone (TSH), both ofwhich are also produced in the pituitary gland, and chorionicgonadotropin (CG), which is produced in the placenta. The hormones arerelatively large (28-38 kilodaltons) and are composed of a common αsubunit non-covalently bound to a distinct β subunit that confersreceptor binding specificity.

The cellular receptors for these hormones are known to be members of theG protein-coupled class of membrane-bound receptors which when activatedstimulate an increase in the activity of adenylyl cyclase. This resultsin an increase in the level of the intracellular second messengeradenosine 3',5'-monophosphate (cAMP), which in turn causes increasedsteroid synthesis and secretion. Hydropathicity plots of the amino acidsequences of these receptors reveal three general domains: (1) ahydrophilic amino-terminal region, considered to be the amino-terminalextracellular domain, (2) seven hydrophobic segments ofmembrane-spanning length, considered to be the transmembrane domain, and(3) a carboxy-terminal region which contains potential phosphorylationsites (serine, threonine, and tyrosine residues), considered to be thecarboxy-terminal intracellular or cytoplasmic domain. The glycoproteinhormone receptor family is distinguished from other G protein-coupledreceptors, such as the β2-adrenergic, rhodopsin, and substance Kreceptors, by the large size of the hydrophilic amino-terminal domain,which is involved in hormone binding.

The FSH receptor is expressed on testicular Sertoli cells and ovariangranulosa cells. While there has been a recognized need for providingessentially pure human FSH receptor, purification of naturally derivedpreparations is not practical and would not likely be sufficient topermit determination of the amino acid sequence. Recently, one group hascloned the cDNA encoding the rat FSH receptor, deduced the amino acidsequence, and expressed it in mammalian cells (Sprengel, Mol.Endocrinol. 4:525, 1990). Another group, attempting to clone the TSHreceptor, apparently also cloned and identified a portion of thetransmembrane region of the human FSH receptor (Parmentier, Science246:1620, 1989).

SUMMARY OF THE INVENTION

The present invention embraces essentially pure human FSH receptor, or afragment or mutant thereof which binds FSH, DNA encoding said receptor,fragment or mutant, expression vectors comprising said DNA, cellstransfected with said expression vectors, and methods of producing saidreceptor, fragment or mutant by culturing said transfected cells. Thepresent invention also includes pharmaceutical compositions comprisingsaid receptor, fragment or mutant, as well as methods of treatingpatients with such compositions to reduce endogenous FSH bioactivity. Animproved assay for human FSH using the receptor, fragment or mutant ofthe present invention is also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of the human FSH receptor cDNA clones. A partialrestriction endonuclease map is shown which was determined by combiningDNA sequence data derived from portions of each of the five clones. Sizeincrements of 0.2 kilobase pair (K) are marked. The regions to which theclones correspond are indicated by the solid lines below the restrictionendonuclease map. The clone designation and the approximate size inkilobase pairs are indicated to the left and right of each solid line,respectively. Approximate locations of the amino-terminal extracellulardomain, transmembrane domain, and carboxy-terminal intracellular domainof the encoded protein are indicated by the dashed arrows above therestriction endonuclease map. The position of an insertion (probably anintron or a portion of an intron) in the 5-10 clone is indicated by theopen box.

FIG. 2 is a strategy for engineering a human FSH receptor DNAconstruction for the purpose of expression of the protein in a mammaliancell line.

FIG. 3 is a map of a plasmid vector useful for the expression of thehuman FSH receptor in mammalian cells.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, human FSH receptor, and its FSH-binding fragments ormutants, refers to polypeptides which are capable of recognizing andselectively binding with human FSH and which produce no significantimmunological response in humans. Thus, the present invention includeshuman FSH receptor having the amino acid sequence depicted in SequenceID No:2, as well as FSH-binding fragments or mutants thereof whichretain a very high degree of homology (i.e. at least 95% identity) withthe depicted amino acid sequence, or with at least the extracellularportion thereof. Fragments of the human FSH receptor embraced by thepresent invention include those fragments remaining after thecytoplasmic and/or the transmembrane domains have been deleted from thefull polypeptide. An especially preferred fragment embraced by thepresent invention is the amino-terminal extracellular portion comprisingapproximately amino acids 1 to 349 of the human FSH receptor amino acidsequence shown in Sequence ID No:2. Since some of the loops which spanthe transmembrane regions (listed in features section, Sequence ID No:2)are also extracellular, these may be linked (e.g. through appropriatespacer molecules) to a fragment containing the amino-terminalextracellular portion to improve binding. The polypeptide may beglycosylated, as in the natural receptor, or may be partially orcompletely deglycosylated.

It is further contemplated that only a portion of the above-describedamino-terminal extracellular domain may be effectively utilized to bindFSH since it is very likely that the complete extracellular domain isnot necessary for this purpose. Thus, a fragment which is somewhatshorter than the 349 amino acids of the complete extracellular domainmay be readily produced and tested for effective binding to FSH. So longas the FSH-binding region of the extracellular portion is maintainedintact, the length of the overall fragment utilized is not critical. Forthis reason, it is also expected that non-interfering amino acids can beadded to either end of the extracellular domain, or FSH-binding fragmentthereof, without adversely affecting the FSH-binding capacity of thepolypeptide. Accordingly, this invention embraces a fragment of thehuman FSH receptor which comprises a substantial portion of theextracellular domain and which retains substantially the sameFSH-binding characteristics as the complete extracellular domain.

Mutant forms of the above-described receptor and fragments are alsowithin the scope of the present invention. Such mutants includeconservative substitutions in one to ten amino acid residues, thelocation and nature of such substitutions being selected so as not tosignificantly degrade the FSH-binding characteristics of the receptor orfragment thereof which is modified.

Essentially pure human FSH receptor is prepared by isolating and cloningthe DNA encoding it from a cDNA or genomic library, ligating the DNAinto a vector, transfecting host cells with the vector, culturingtransfected host cells under conditions which permit expression of thereceptor, fragment or mutant, and recovering the receptor, fragment ormutant from the culture.

The DNA which is used to make expression vectors may be genomic DNA orcDNA encoding human FSH receptor, and may contain regions which enhanceexpression, such as introns, promoters, enhancers, etc. The DNA may bereadily modified by substitution, deletion or insertion of nucleotides(e.g. by site-specific mutagenesis) that do not adversely affect thebiological or FSH-binding activity of the expressed protein. Forexample, conservative substitutions (mutations) which alter from one toten amino acids may be made without adversely affecting the overallstructure and activity of the expressed protein (mutein). Likewise,certain portions of the DNA, such as those portions which code for thecytoplasmic and/or transmembrane domains, may be deleted so that only afragment, such as the soluble extracellular domain, of the protein isexpressed. The human FSH receptor or FSH-binding fragment or mutantthereof may also be expressed as a fusion protein. One such fusionprotein would include a polypeptide at the carboxy-terminus which wouldconfer characteristics which would facilitate purification of theprotein, or immobilization of the purified protein on a solid substratefor use in FSH assays or FSH purification protocols. Another such fusionprotein would include a cleavable polypeptide at the amino-terminuswhich would facilitate expression.

Human FSH receptor produced in accordance with the present invention isessentially pure, meaning that it is substantially free of biologicaladventitious agents normally associated with FSH receptor extracted fromnatural sources, such as, for example, bacteria, viruses, and otherproteins. The receptor may be formulated into pharmaceuticalcompositions by mixing with suitable pharmaceutically acceptablecarriers in a manner known to those skilled in the art.

Generally, pharmaceutical compositions may be formulated for oral,parenteral (including subcutaneous, intramuscular, and intravenous),vaginal, rectal, buccal (including sublingual), transdermal orintranasal administration. Compositions for parenteral administrationare normally in the form of a liquid solution, dispersion, or emulsion,preferably isotonic; for vaginal or rectal administration, as a cream orsuppository; for oral or buccal administration, as a tablet or capsule;and for intranasal administration, as a powder, nasal drop or aerosol.Various slow release, depot implant or injectable dosage forms may beutilized. The active component may also be incorporated into polymermatrices, liposomes and microspheres to control delivery.

These compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art. Formulations for parenteral administration maycontain as common excipients sterile water or saline, alkylene glycolssuch as propylene glycol, polyalkylene glycols such as polyethyleneglycol, oils of vegetable origin, hydrogenated naphthalenes and thelike. Formulations for vaginal or rectal administration, e.g.suppositories, may contain as excipients, for example,polyalkyleneglycols, vaseline, cocoa butter, and the like. Formulationsfor nasal administration may be in powder form and contain asexcipients, for example, lactose or dextran, or may be aqueous or oilysolutions for administration in the form of nasal drops or meteredspray. For buccal administration, typical excipients include sugars,calcium stearate, pregelatinated starch and the like. One or moresurfactant acids or salts can be added to the solution or powderformulation. Suitable pharmaceutically acceptable surfactant salts willbe those which retain the phenomenon of enhanced peptide absorption, aswell as the compound's surfactant characteristics and which are notdeleterious to the subject or otherwise contraindicated.

The dosage of active ingredient administered, as well as the route andfrequency of administration, will obviously depend upon the needs andcondition of the patient being treated, the therapeutic effect which issought, and the judgement of the doctor conducting the treatment.

Pharmaceutical compositions comprising human FSH receptor, orFSH-binding fragments or mutants thereof, may be administered to apatient in therapeutically effective doses to bind with endogenouscirculating FSH in the patient and thereby control the available levelof bioactive FSH. Thus, pharmaceutical compositions of the presentinvention are effectively utilized to reduce endogenous FSH bioactivity.In a female patient, such treatment may be effectively used to preventfollicle growth and maturation, thereby preventing pregnancy. In a malepatient, such treatment may be effectively used to preventspermatogenesis. A particularly suitable pharmaceutical composition forthe above purpose comprises a fragment of the human FSH receptor whichcomprises the amino-terminal extracellular domain or a substantialportion thereof with substantially the same FSH-binding characteristics.

Essentially pure human FSH receptor may also be advantageously utilizedin conventional receptor assays for FSH, such as, for example, thatdisclosed in Reichert, Endocrinolgy 94:483, 1974. Substitution of thepure receptor of the present invention will substantially improve theconsistency and performance of such assays. The receptor can also beincorporated into a stable cell line, particularly a mammalian cellline, capable of producing a measurable biological response uponstimulation of the receptor. Measurement of cell response in thepresence of FSH under assay (e.g. in test serum, plasma, culture media,tissue homogenates, etc.) would provide an indication of bioactivity,and thereby provide a highly useful diagnostic assay. Such a cell linecould also be used to screen chemical libraries for substances that mayinteract with the FSH receptor or to test peptides or small proteins fortheir ability to bind to the FSH receptor in a rapid through-putscreening system. An example of a rapid through-put screening system maybe one in which the binding of a ligand to the recombinant FSH receptorresults in the generation of cAMP which activates the luciferase geneoperatively linked to a cAMP response element and can be quantitated bythe measurement of bioluminescence. A fragment comprising theamino-terminal extracellular domain or an FSH-binding fragment and/orfusion protein thereof can be linked to an affinity column to purify FSHfrom fluids, extracts, etc.

Essentially pure human FSH receptor or FSH-binding fragments or mutantsthereof can also be used in X-ray crystallographic analysis to developmolecular models. Such models are useful in defining the tertiarystructure of the hormone-binding domains of the human FSH receptor. Suchinformation would provide insight into the structure of the actualregions of contact between FSH and its receptor, thus aiding the designof peptides which have FSH agonistic or antagonistic activity.

The recombinant techniques suitable for producing the proteins and DNAof the present invention, including identification of suitable mutationtechniques, vectors, host cells, culture conditions, etc., arewell-known to those skilled in the art and are adequately described, forexample, in U.S. Pat. No. 4,761,371 and WO 88/09818, the disclosures ofwhich are incorporated herein by reference. The experimental protocols,bacterial and bacteriophage culture media and chemical solutions used inthe examples below are described in detail in Sambrook, "MolecularCloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor LaboratoryPress, 1989, unless otherwise referenced.

EXAMPLE 1 Isolation and Characterization of Human FSH Receptor cDNAClones

Library screening

A rat FSH receptor cDNA clone similar to that described by Sprengel,Mol. Endocrinol. 4:525, 1990 was obtained from Dr. William Moyle,University of Medicine and Dentistry of New Jersey-Robert Wood JohnsonMedical School. This cDNA clone was inserted into the SV40 lateexpression vector pVSL (Pharmacia LKB, product number 27-4509-01) andwas designated pSVLFSHR. A 2.1 kilobase pair (kb) DNA fragmentcontaining the region corresponding to the complete rat FSH receptorprotein coding region was excised from the plasmid using the restrictionendonuclease sites XbaI and BamHI. The digested DNA wassize-fractionated by gel electrophoresis, and the 2.1 kb rat FSHreceptor fragment was purified by electroelution from the gel. Thispurified DNA fragment was used as a probe for library screening toidentify human FSH receptor cDNA clones.

A lambda gt11 cDNA library, constructed from RNA extracted from humantestis, was purchased from Clontech, Palo Alto, Calif. (catalogue numberHL1010b) and was amplified prior to use. Twenty aliquots of theamplified library, equivalent to approximately 7.5×10⁴ plaque formingunits (pfu), were each adsorbed to about 0.5 milliliters (ml) of aplating suspension of E. coli strain Y1088. The suspension had beenprepared by growing an overnight culture of Y1088 at 37° C. in NZYM orLB supplemented with 0.2% maltose and 10 millimolar (mM) MgSO₄,pelleting the cells, and then resuspending them in 10 mM MgSO₄ to anO.D.₆₀₀ of 0.5. About 6.5 ml of molten NZYM top agarose (0.7%), at atemperature of 48° C., was added to each phage/cell suspension and theresulting mixture was poured onto one of twenty 150 millimeter (mm) NZYMagar plates (prewarmed to 42° C.). The total number of phage plated forthe primary screen was about 1.5×10⁶. After a 4 hour incubation at 42°C., followed by chilling at 4° C. for several hours, duplicatenitrocellulose filter (Millipore) plaque lifts were generated from eachplate according to the procedures of Benton and Davies (Science,196:180, 1977). The random oligonucleotide priming procedure of Feinbergand Vogelstein (Anal. Biochem. 137:266, 1983) was used to generate, fromthe template rat FSH receptor DNA fragment described in the precedingparagraph, a ³² P-labeled probe of a specific activity of 1-2×10⁹ countsper minute (cpm)/microgram (μg). Prehybridization of the nitrocellulosefilter phage lifts was done in a buffer containing 50% formamide, 5× SSC[1× SSC is 0.15 molar (M) sodium chloride, 0.015 M sodium citrate], 20mM sodium phosphate buffer, pH 7.2, 10× Denhardt's reagent(50×Denhardt's reagent is 1% Ficoll, 1% polyvinylpyrrolidone and 1%bovine serum albumin) and 100 μg/ml tRNA at 37° C. for about 6 hours.Hybridization of the filters was done in the same buffer except that therat FSH receptor DNA probe was added at a concentration of about 3×10⁶cpm/ml buffer. After hybridization for 16-24 hours at 37° C., excessprobe was washed from the filters in 2× SSC, 0.1% SDS at roomtemperature for 30 minutes, then 0.2× SSC, 0.1% SDS at 37° C. for 60minutes. The filters were subsequently exposed to XAR film (Kodak)overnight at -70° C. Six duplicate positives were identified from theprimary library screen. With the wide end of a pasteur pipette,plaque-containing agar plugs were removed from the 150-mm plates in theregions in which the positive clones were located. The phage were elutedby soaking the plugs in SM. The suspended phage were then replated onto150-mm NZYM agar plates as described for the primary screen. Plateswhich contained about 500 plaque forming units per plate were selectedfor secondary screening. The procedures used for filter lifts and filterhybridizations for the secondary screen were the same as described forthe primary screen.

Following the secondary screen, 5 putative human FSH receptor positiveswere identified and isolated as purified λgt11 bacteriophage clones.They were assigned the following designations: 1-5, 5-10, 11-11, 13-9,and 15-6.

Determination of the DNA Sequences of the Putative Human FSH ReceptorcDNA Clones

Bacteriophage DNA was prepared from each of the λgt11 cDNA isolatesusing the plate lysate method described in Sambrook, "Molecular Cloning:A Laboratory Manual," 2nd ed., Cold Spring Harbor Laboratory Press,1989, page 2.118. The bacteriophage DNA was digested with EcoRIrestriction endonuclease, and then size-fractionated in an agarose gelfor purification of the cDNA insert fragments. The purified cDNA insertswere subcloned into the EcoRI site of pUC18 in order to facilitatesubsequent cloning and sequencing manipulations. Double-stranded plasmidDNA was purified from 5 ml cultures of the host strain E. coli MC1061 bythe small-scale alkaline lysis method (Sambrook, ibid. p. 1.25), thenfurther purified by passage over Elutip-d columns (Schleicher & Schuell,Keene, NH) using the manufacturer's protocol. Half of the plasmid DNAobtained from each small-scale plasmid preparation was then denatured in0.2 normal (N) NaOH, 0.2 mM EDTA in a volume of 20 microliters (μl) atroom temperature for 10 minutes. The denatured plasmid DNA wasneutralized and ethanol precipitated by adding 7.5 μl of 7.5 M ammoniumacetate and 110 μl 100% ethanol and chilling the mixture in liquidnitrogen. The DNA precipitate was pelleted by centrifugation in amicrofuge for 10 minutes. The DNA pellets were washed in 70% ethanol anddried. Sequencing reactions were done with Sequenase T7 DNA polymerase(United States Biochemical) according to the manufacturer'sspecifications. Preliminary sequencing reactions were done with forwardand reverse sequencing primers (Pharmacia LKB) for the pUC18 polylinkerregion. Data generated from preliminary sequencing was used to designhuman FSH receptor-specific sequencing primers. The primers were eithersynthesized on a model 391 Applied Biosystems DNA synthesizer or orderedfrom National Biosciences, Inc., Hamel, Minn. Some DNA sequence data wasobtained by subcloning smaller restriction endonuclease fragments of theoriginal clones into pUC18 and repeating sequencing reactions with theforward and reverse primers.

Preliminary sequence data showed that none of the five cDNA isolatesrepresented the full-length protein coding region of the human FSHreceptor, but that the combined sequence data from the clones could beused to infer the complete protein sequence. A schematic diagram of therelative locations of each of the five clones in relation to a map ofthe complete human FSH receptor cDNA sequence is shown in FIG. 1. Thecomplete human FSH receptor cDNA sequence, obtained by combining thesequence readings from the overlapping clones using the GeneticsComputer Group (GCG) fragment assembly computer program, is depicted inSequence ID No:1 and the inferred amino acid sequence is depicted inSequence ID No:2. Analysis of the human FSH receptor DNA sequenceresulted in the identification of a long open reading frame of 2085nucleotides which encoded a protein of 695 amino acids. The human FSHreceptor is therefore 3 amino acids longer than the rat FSH receptor.The overall percent identities between the rat and human FSH receptorDNA and protein sequences were determined using the GCG Bestfit programand were 86% and 89%, respectively. The extracellular amino-terminalhydrophilic portion of the human FSH receptor is estimated to be 349amino acids in length, and shares 87% identity with the correspondingregion of the rat FSH receptor. The seven membrane spanning regions ofthe two species, which are bridged by three extracellular and threeintracellular loops, share 95% identity, and the carboxy-terminalintracellular regions share only 81% identity. The partial amino acidsequence published by Parmentier, Science 246:1620-1622, 1989,corresponds to amino acids 399 to 525 in Sequence ID No:2.

Clone 5-10 was a variant in that it contained an insertion of 0.25 kbafter the T at nucleotide position 448 in Sequence ID No:1. The DNAsequence of the insertion was not similar to any portion of the rat orhuman FSH receptor DNA sequence and also was not similar to any knownsequence in the Genbank or EMBL DNA sequence databases. Thecorresponding region in clone 11-11 did not contain this insertion. LHreceptor cDNA clones isolated from a human thyroid cDNA library containsimilar variations (Frazier, Mol. Endocrinol. 4:1264-1276, 1990). Thesevariants are likely derived from incompletely and/or aberrantly splicedmRNA molecules. The presence of a 3' splice consensus sequence (CAG'G)at the 3' junction of the 5-10 insertion is evidence which supports thisexplanation.

The pUC18 plasmids harboring the 11-11 (called pHFSHR11-11), 15-6(called pHFSHR15-6), and 5-10 (called pHFSHR5-10) cDNA inserts weredeposited in the American Type Culture Collection (ATCC), Rockville,Md., on Mar. 1, 1991 and have received the accession numbers ATCC68538,ATCC68540, and ATCC68539, respectively. These deposits were made inaccordance with all of the requirements of the Budapest Treaty.

EXAMPLE 2 Expression of Human FSH Receptor in Mammalian Cells andDemonstration of Bioactivity

Construction of Vectors for the Expression of the Full-length Human FSHReceptor in Mammalian Cells

The strategy for the engineering a human FSH receptor DNA construct forexpression in mammalian cells is shown in FIG. 2. The 5' 691 base pair(bp) NsiI-BamHI fragment which contains the start ATG is purified frompHFSHR1-11 and subcloned into pUC18 digested with PstI and BamHI. Theresulting plasmid, pHFSHR11-11nb, is linearized with SphI. The terminiare blunted by treatment with T4 polymerase or the Klenow fragment ofDNA polymerase I (New England Biolabs, Beverly, Mass.). After ligationof XhoI linkers (New England Biolabs, Beverly, Mass.) to the bluntedtermini, the mixture is digested with XhoI and BamHI and theapproximately 700 bp human FSH receptor 5' fragment is gel purified. Afragment from the middle region of the human FSH receptor CDNA isisolated from pHFSHR11-11 by digestion with BamHI and SphI and gelpurification of the appropriate 734 bp piece. The 3' human FSH receptorfragment, which contains the TAA stop codon, is isolated from pHFSHR15-6by first digesting the plasmid with DraIII, then blunting the termini ina reaction with T4 DNA polymerase or the Klenow fragment of E. coli DNApolymerase I. XhoI linkers are ligated to the blunted ends and theresulting mixture is digested with SphI and XhoI and thensize-fractionated so that the 3' 690 bp (approximately) SphI-XhoIfragment is isolated and purified. The complete human FSH receptorexpression construction is assembled, with the 5' XhoI-BamHI, middleBamHI-SphI, and 3' SphI-XhoI human FSH receptor fragments, as a 2.1 kbXhoI fragment in pUC-XhoI (created in this laboratory by converting theSmaI site in the pUC18 polylinker to an XhoI site with XhoI linkers).Correct assembly of the construction is verified by restrictionendonuclease digestion and DNA sequencing. The human FSH receptorexpression construction is then purified as a 2.1 kb XhoI fragment andinserted into the XhoI site of an expression vector so that a plasmidsuch as the one depicted in FIG. 3 is obtained. If the expression vectorselected does not have a suitable XhoI cloning site, linkers can be usedto convert either the fragment termini or the vector cloning site intocompatible sites. Transcription of the human FSH receptor coding regionand the marker gene (for selection and/or amplification) is initiated bya eukaryotic promoter such as the mouse metallothoinein I (MMT-I), RousSarcoma Virus (RSV) or simian virus 40 early or late (SV40E, SV40L)promoters. Suitable marker genes are the neomycin resistance gene (Neo),the dihydrofolate reductase (DHFR) gene, and the multidrug resistancegene (MDR). A polyadenylation signal (poly A), usually the SV40 earlypolyadenylation region, is also provided for the processing of thereceptor gene and the marker gene transcripts. The expression vectoralso includes pML, a derivative of pBR322 which contains the ampicillinresistance gene and a bacterial origin of replication, to enable growthand propagation of the plasmid in a suitable E. coli strain.

Similarly, DNA cloning and engineering techniques can be used by thoseskilled in the art to modify the human FSH receptor DNA expressionconstruction so that FSH-binding fragments are encoded. These modifiedDNA fragments can be inserted into an expression vector similar to theone shown in FIG. 3 and used to transfect mammalian cells to developlines which secrete soluble FSH-binding fragments of the human FSHreceptor.

Demonstration of Bioactivity by Transient Transfection of COS-7 Cellswith the Human FSH Receptor-containing Expression Vector

To test the hormone mediated activation of the recombinant human FSHreceptor, the human FSH receptor expression vector construct istransfected into COS-7 cells which are stimulated with FSH to elicit abiological response measured by an increase in the amount ofintracellular cAMP levels.

The vector plasmid DNA is purified by two sequential high-speedcentrifugations through cesium chloride density gradients. Exponentiallygrowing COS-7 cells (ATCC CRL 1651) are transfected with the DNA using amodification of the DEAE-dextran transfection protocol of Seed andAruffo (PNAS 84:3366, 1987). COS-7 cells are seeded in growth medium insix well plates at a density of 1×10⁵ cells per well. Growth medium forCOS-7 cells is Dulbecco's modified Eagle's medium (DMEM) supplementedwith 10% fetal bovine serum (FBS) and 1% L-glutamine. After 16 to 25hours incubation at 37° C., the medium is removed and the cells washedtwice with phosphate-buffered saline (PBS). A DNA mixture containing thehuman FSH expression vector plasmid, or a control vector plasmid whichdoes not include the human FSH receptor cDNA insert, is added dropwiseto the center of the culture in each well. The dishes are swirled gentlyto distribute the mixtures evenly. The DNA mixture for each well isprepared by the following method: (1) 2 μg vector plasmid DNA is dilutedin PBS so that the total volume is 136 μl, (2) 68 μl of a solution of 2mg/ml DEAE-dextran (Pharmacia LKB), 0.9% NaCl is diluted in 68 μl PBS,and (3) the solutions made in (1) and (2) are combined to obtain a DNAmixture in a total volume of 272 μl. Mock-transfected control cells aretreated with the same solution expect that DNA is omitted. After a 15minute incubation at 37° C., the DNA or mock mixture is gently removedand the cells are washed twice with PBS. Growth medium which alsocontains 100 micromolar (μM) chloroquine diphosphate (Sigma) is addedand the cells are left to incubate at 37° C. for 3 hours. The medium isremoved, the cells are washed with DMEM and then incubated at 37° C. for48 hours in growth medium.

Each well of cells is subsequently washed with 3 ml of growth medium andthen pre-incubated in 700 μl of DMEM (without FBS) containing 0.1 mM3-isobutyl-1-methylxanthine (Sigma) for 15 minutes at 37° C. Followingpre-incubation, purified human FSH or LH is added to the medium in eachwell and incubation is continued for an additional 30 min at 37° C. Theassay is then terminated by four cycles of rapid freezing and thawing.50 μl of cell lysate is removed for protein determination. To theremaining lysate, which is to be processed for cAMP determination, 840μl of ice cold ethanol is added. Both aliquots are centrifuged at13,000×g for 15 minutes to remove cell debris. Determination of thetotal protein content in each 50 μl aliquot of cell lysate sample iscarried out using the Bio-Rad Protein Assay kit (catalogue No.500-0002).

cAMP assays are done using the RIANEN cAMP (¹²⁵ I) RIA kit, cataloguenumber NEK-033, purchased from Dupont/NEN Medical Products, Boston,Mass. The solutions used in the following protocol are provided in thekit. 200 μl of the hormonally stimulated or non-stimulated cell lysateis dried-down and resuspended in cAMP assay buffer. The samples areacetylated and brought to a final volume of 1 ml. 100 μl of samples anda set of acetylated cAMP standards are added to 100 μl of tracer cAMPsolution followed by the addition of 100 μl antiserum complex. Themixtures are incubated at 4° C. for overnight. The mixtures areprecipitated with 500 μl of precipitator solution, centrifuged at 1200×gfor 15 minutes. The supernatant is removed and the activity remaining inthe pellets is measured in a Packard Cobra auto gamma counter.

If a suitable range of FSH concentrations is used for stimulation of thehuman FSH receptor-expressing cells, such as, for example, 0.01 to 1000nanograms per milliliter, a CAMP dose response curve can be generatedwhich shows a proportional increase in cAMP levels as increased amountsof FSH are used for cell stimulation.

EXAMPLE 3 Development of Mammalian Cell Lines which Stably ExpressRecombinant Human FSH Receptor or an FSH-binding Fragment or MutantThereof

Suitable mammalian cell lines for expression of recombinant human FSHand its derivatives include Chinese Hamster Ovary (CHO), mouseadenocarcinoma Y1, rat pituitary GH₃, human breast carcinoma MCF7, andhuman embryonic kidney 293. In this example, the use of Y1 and CHO cellsis described.

Y1 cells are a clonal steroid secreting cell strain initiated from amouse adrenal cortex tumor (Yasumura, Cancer Res. 26: 529-536, 1966.).These cells were obtained from the ATCC cell bank (ATCC CCL 79) and aremaintained in culture by growth in Ham's F10 medium supplemented with15% horse serum (HS), 2.5% FBS, and 1% L-glutamine (Y1 growth medium).

CHO-DUKX cells are a clonal mutant of Chinese hamster ovary cellslacking dihydrofolate reductase activity (Urlaub G. and Chasin, L. A.PNAS 77: 4216-4220, 1980). The cells were maintained in MinimumEssential Alpha Medium (MEM-α) supplemented with 10% FBS and 1%L-glutamine (CHO growth medium).

Calcium Phosphate Transfections

24 hours prior to transfection, cells are plated on 100-mm dishes.CHO-DUKX cells are plated at a density of 7×10⁵ cells/dish, Y1 cells areplated at a density of 1×10⁶ cells/dish. 10 μg of the vector plasmid DNAsuch as the one depicted in FIG. 3 is added to 0.5 ml of transfectionbuffer. The buffer is prepared by adding 4 grams (g) NaCl, 0.185 g KCl,0.05 g Na₂ HPo4, 0.5 g dextrose, 2.5 g Hepes and sterile, distilled H₂ Oto a final volume of 500 ml and adjusting the pH to 7.5. 31 μl of 2 MCaCl₂ is added to the DNA/transfection buffer mixture and vortexed. Thissolution is allowed to stand at room temperature for 45 minutes (Graham,Virology 52: 456, 1973). After 45 minutes the media is removed from thecells and the DNA-CaCl₂ -transfection buffer precipitate is layered overthe cells. The cells are allowed to stand at room temperature for 20minutes, after which 5 ml of the appropriate growth medium is added andthe plates are incubated for 6 hours at 370°. After 6 hours, the cellsare shocked by aspirating off the media and adding 5 ml of transfectionbuffer containing 15% glycerol for 3.5 minutes. The cells are rinsedtwice with PBS prior to the addition of 10 ml growth medium. 48 hourspost transfection the CHO-DUKX cells are subcultured at a 1:10 splitratio and selection medium is added. Y1 cells are allowed to grow for 72hours post transfection before subculturing at a 1:5 split ratio inselection medium. Selection medium for DHFR in CHO-DUKX cells isprepared by adding MEM-α without ribonucleosides and ribonucleosidessupplemented with 10% dialyzed FBS and 1% L-glutamine+0.02 μMmethotrexate (MTX). Selection medium for Neo in Y1 cells consists of Y1growth medium+80 μg/ml G418.

Determination of FSH in vitro Bioactivity Using Y1 or CHO Cells StablyTransfected with the Human FSH Receptor

In order to estimate the bioactivity of pharmaceutical preparations ofFSH, CHO cells which stably express the human FSH receptor can betreated with FSH and assayed for increased cAMP levels as describedabove for COS-7 cells in example 2. Alternatively, a reporter gene suchas luciferase, which may or may not be secreted by the cell, can beoperatively linked to a cAMP response element, and increases in cAMPlevels may be indirectly measured by a non-radioactive method, such asbioluminescence. If the DHFR gene was used for selection of transfectedcells, the human FSH receptor content of the cell can be increased byexposing the cells to stepwise increases of MTX, so that the vector copynumber in increased. This can be done, if necessary, to adjust thebiological response to an optimal range for the assay.

Y1 cells which stably express the human FSH receptor can be treated withFSH and the culture medium assayed for increased levels of progesterone.A kit such as the Serono Diagnostics Progesterone MAIA, product number12274, distributed by Ciba Corning, Medfield, Mass., can be used forthis purpose.

Mammalian cells which express high levels of human FSH receptor orFSH-binding fragments or mutants thereof, can be used for themanufacture of large amounts of human FSH receptor protein for use inreceptor radioimmunoassays.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  2                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  2179                                                             (B) TYPE:  Nucleic a - #cid                                                   (C) STRANDEDNESS:  Doub - #le                                                 (D) TOPOLOGY:  Linear                                                - -     (ii) MOLECULE TYPE:  cDNA to mRNA                                     - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM:  Homo - #sapiens                                                (F) TISSUE TYPE:  Te - #stis                                         - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY:  lgt11 - #cDNA library, ClonTech #HL1010b                        (B) CLONE:  pHFSHR11-11 - #, pHFSHR15-6                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: protein c - #oding region                                       (B) LOCATION: 75 to - #2159                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #1:                          - - TGTGGAGCTT CTGAGATCTG TGGAGGTTTT TCTCTGCAAA TGCAGGAAGA AA -             #TCAGGTGG     60                                                                 - - ATGGATGCAT AATT ATG GCC CTG CTC CTG GTC TCT T - #TG CTG GCA TTC        CTG      110                                                                                    Met A - #la Leu Leu Leu Val Ser Leu Leu Ala Phe Le - #u                        - #      -15           - #      -10                          - - AGC TTG GGC TCA GGA TGT CAT CAT CGG ATC TG - #T CAC TGC TCT AAC AGG          158                                                                       Ser Leu Gly Ser Gly Cys His His Arg Ile Cy - #s His Cys Ser Asn Arg            -5                 - # 1               5  - #                 10              - - GTT TTT CTC TGC CAA GAG AGC AAG GTG ACA GA - #G ATT CCT TCT GAC CTC          206                                                                       Val Phe Leu Cys Gln Glu Ser Lys Val Thr Gl - #u Ile Pro Ser Asp Leu                        15     - #             20     - #             25                  - - CCG AGG AAT GCC ATT GAA CTG AGG TTT GTC CT - #C ACC AAG CTT CGA GTC          254                                                                       Pro Arg Asn Ala Ile Glu Leu Arg Phe Val Le - #u Thr Lys Leu Arg Val                    30         - #         35         - #         40                      - - ATC CAA AAA GGT GCA TTT TCA GGA TTT GGG GA - #C CTG GAG AAA ATA GAG          302                                                                       Ile Gln Lys Gly Ala Phe Ser Gly Phe Gly As - #p Leu Glu Lys Ile Glu                45             - #     50             - #     55                          - - ATC TCT CAG AAT GAT GTC TTG GAG GTG ATA GA - #G GCA GAT GTG TTC TCC          350                                                                       Ile Ser Gln Asn Asp Val Leu Glu Val Ile Gl - #u Ala Asp Val Phe Ser            60                 - # 65                 - # 70                 - # 75       - - AAC CTT CCC AAA TTA CAT GAA ATT AGA ATT GA - #A AAG GCC AAC AAC CTG          398                                                                       Asn Leu Pro Lys Leu His Glu Ile Arg Ile Gl - #u Lys Ala Asn Asn Leu                            80 - #                 85 - #                 90              - - CTC TAC ATC AAC CCT GAG GCC TTC CAG AAC CT - #T CCC AAC CTT CAA TAT          446                                                                       Leu Tyr Ile Asn Pro Glu Ala Phe Gln Asn Le - #u Pro Asn Leu Gln Tyr                        95     - #            100     - #            105                  - - CTG TTA ATA TCC AAC ACA GGT ATT AAG CAC CT - #T CCA GAT GTT CAC AAG          494                                                                       Leu Leu Ile Ser Asn Thr Gly Ile Lys His Le - #u Pro Asp Val His Lys                   110          - #       115          - #       120                      - - ATT CAT TCT CTC CAA AAA GTT TTA CTT GAC AT - #T CAA GAT AAC ATA AAC          542                                                                       Ile His Ser Leu Gln Lys Val Leu Leu Asp Il - #e Gln Asp Asn Ile Asn               125              - #   130              - #   135                          - - ATC CAC ACA ATT GAA AGA AAT TCT TTC GTG GG - #G CTG AGC TTT GAA AGT          590                                                                       Ile His Thr Ile Glu Arg Asn Ser Phe Val Gl - #y Leu Ser Phe Glu Ser           140                 1 - #45                 1 - #50                 1 -      #55                                                                              - - GTG ATT CTA TGG CTG AAT AAG AAT GGG ATT CA - #A GAA ATA CAC AAC        TGT      638                                                                    Val Ile Leu Trp Leu Asn Lys Asn Gly Ile Gl - #n Glu Ile His Asn Cys                          160  - #               165  - #               170              - - GCA TTC AAT GGA ACC CAA CTA GAT GAG CTG AA - #T CTA AGC GAT AAT AAT          686                                                                       Ala Phe Asn Gly Thr Gln Leu Asp Glu Leu As - #n Leu Ser Asp Asn Asn                       175      - #           180      - #           185                  - - AAT TTA GAA GAA TTG CCT AAT GAT GTT TTC CA - #C GGA GCC TCT GGA CCA          734                                                                       Asn Leu Glu Glu Leu Pro Asn Asp Val Phe Hi - #s Gly Ala Ser Gly Pro                   190          - #       195          - #       200                      - - GTC ATT CTA GAT ATT TCA AGA ACA AGG ATC CA - #T TCC CTG CCT AGC TAT          782                                                                       Val Ile Leu Asp Ile Ser Arg Thr Arg Ile Hi - #s Ser Leu Pro Ser Tyr               205              - #   210              - #   215                          - - GGC TTA GAA AAT CTT AAG AAG CTG AGG GCC AG - #G TCG ACT TAC AAC TTA          830                                                                       Gly Leu Glu Asn Leu Lys Lys Leu Arg Ala Ar - #g Ser Thr Tyr Asn Leu           220                 2 - #25                 2 - #30                 2 -      #35                                                                              - - AAA AAG CTG CCT ACT CTG GAA AAG CTT GTC GC - #C CTC ATG GAA GCC        AGC      878                                                                    Lys Lys Leu Pro Thr Leu Glu Lys Leu Val Al - #a Leu Met Glu Ala Ser                          240  - #               245  - #               250              - - CTC ACC TAT CCC AGC CAT TGC TGT GCC TTT GC - #A AAC TGG AGA CGG CAA          926                                                                       Leu Thr Tyr Pro Ser His Cys Cys Ala Phe Al - #a Asn Trp Arg Arg Gln                       255      - #           260      - #           265                  - - ATC TCT GAG CTT CAT CCA ATT TGC AAC AAA TC - #T ATT TTA AGG CAA GAA          974                                                                       Ile Ser Glu Leu His Pro Ile Cys Asn Lys Se - #r Ile Leu Arg Gln Glu                   270          - #       275          - #       280                      - - GTT GAT TAT ATG ACT CAG ACT AGG GGT CAG AG - #A TCC TCT CTG GCA GAA         1022                                                                       Val Asp Tyr Met Thr Gln Thr Arg Gly Gln Ar - #g Ser Ser Leu Ala Glu               285              - #   290              - #   295                          - - GAC AAT GAG TCC AGC TAC AGC AGA GGA TTT GA - #C ATG ACG TAC ACT GAG         1070                                                                       Asp Asn Glu Ser Ser Tyr Ser Arg Gly Phe As - #p Met Thr Tyr Thr Glu           300                 3 - #05                 3 - #10                 3 -      #15                                                                              - - TTT GAC TAT GAC TTA TGC AAT GAA GTG GTT GA - #C GTG ACC TGC TCC        CCT     1118                                                                    Phe Asp Tyr Asp Leu Cys Asn Glu Val Val As - #p Val Thr Cys Ser Pro                          320  - #               325  - #               330              - - AAG CCA GAT GCA TTC AAC CCA TGT GAA GAT AT - #C ATG GGG TAC AAC ATC         1166                                                                       Lys Pro Asp Ala Phe Asn Pro Cys Glu Asp Il - #e Met Gly Tyr Asn Ile                       335      - #           340      - #           345                  - - CTC AGA GTC CTG ATA TGG TTT ATC AGC ATC CT - #G GCC ATC ACT GGG AAC         1214                                                                       Leu Arg Val Leu Ile Trp Phe Ile Ser Ile Le - #u Ala Ile Thr Gly Asn                   350          - #       355          - #       360                      - - ATC ATA GTG CTA GTG ATC CTA ACT ACC AGC CA - #A TAT AAA CTC ACA GTC         1262                                                                       Ile Ile Val Leu Val Ile Leu Thr Thr Ser Gl - #n Tyr Lys Leu Thr Val               365              - #   370              - #   375                          - - CCC AGG TTC CTT ATG TGC AAC CTG GCC TTT GC - #T GAT CTC TGC ATT GGA         1310                                                                       Pro Arg Phe Leu Met Cys Asn Leu Ala Phe Al - #a Asp Leu Cys Ile Gly           380                 3 - #85                 3 - #90                 3 -      #95                                                                              - - ATC TAC CTG CTG CTC ATT GCA TCA GTT GAT AT - #C CAT ACC AAG AGC        CAA     1358                                                                    Ile Tyr Leu Leu Leu Ile Ala Ser Val Asp Il - #e His Thr Lys Ser Gln                          400  - #               405  - #               410              - - TAT CAC AAC TAT GCC ATT GAC TGG CAA ACT GG - #G GCA GGC TGT GAT GCT         1406                                                                       Tyr His Asn Tyr Ala Ile Asp Trp Gln Thr Gl - #y Ala Gly Cys Asp Ala                       415      - #           420      - #           425                  - - GCT GGC TTT TTC ACT GTC TTT GCC AGT GAG CT - #G TCA GTC TAC ACT CTG         1454                                                                       Ala Gly Phe Phe Thr Val Phe Ala Ser Glu Le - #u Ser Val Tyr Thr Leu                   430          - #       435          - #       440                      - - ACA GCT ATC ACC TTG GAA AGA TGG CAT ACC AT - #C ACG CAT GCC ATG CAG         1502                                                                       Thr Ala Ile Thr Leu Glu Arg Trp His Thr Il - #e Thr His Ala Met Gln               445              - #   450              - #   455                          - - CTG GAC TGC AAG GTG CAG CTC CGC CAT GCT GC - #C AGT GTC ATG GTG ATG         1550                                                                       Leu Asp Cys Lys Val Gln Leu Arg His Ala Al - #a Ser Val Met Val Met           460                 4 - #65                 4 - #70                 4 -      #75                                                                              - - GGC TGG ATT TTT GCT TTT GCA GCT GCC CTC TT - #T CCC ATC TTT GGC        ATC     1598                                                                    Gly Trp Ile Phe Ala Phe Ala Ala Ala Leu Ph - #e Pro Ile Phe Gly Ile                          480  - #               485  - #               490              - - AGC AGC TAC ATG AAG GTG AGC ATC TGC CTG CC - #C ATG GAT ATT GAC AGC         1646                                                                       Ser Ser Tyr Met Lys Val Ser Ile Cys Leu Pr - #o Met Asp Ile Asp Ser                       495      - #           500      - #           505                  - - CCT TTG TCA CAG CTG TAT GTC ATG TCC CTC CT - #T GTG CTC AAT GTC CTG         1694                                                                       Pro Leu Ser Gln Leu Tyr Val Met Ser Leu Le - #u Val Leu Asn Val Leu                   510          - #       515          - #       520                      - - GCC TTT GTG GTC ATC TGT GGC TGC TAT ATC CA - #C ATC TAC CTC ACA GTG         1742                                                                       Ala Phe Val Val Ile Cys Gly Cys Tyr Ile Hi - #s Ile Tyr Leu Thr Val               525              - #   530              - #   535                          - - CGG AAC CCC AAC ATC GTG TCC TCC TCT AGT GA - #C ACC AGG ATC GCC AAG         1790                                                                       Arg Asn Pro Asn Ile Val Ser Ser Ser Ser As - #p Thr Arg Ile Ala Lys           540                 5 - #45                 5 - #50                 5 -      #55                                                                              - - CGC ATG GCC ATG CTC ATC TTC ACT GAC TTC CT - #C TGC ATG GCA CCC        ATT     1838                                                                    Arg Met Ala Met Leu Ile Phe Thr Asp Phe Le - #u Cys Met Ala Pro Ile                          560  - #               565  - #               570              - - TCT TTC TTT GCC ATT TCT GCC TCC CTC AAG GT - #G CCC CTC ATC ACT GTG         1886                                                                       Ser Phe Phe Ala Ile Ser Ala Ser Leu Lys Va - #l Pro Leu Ile Thr Val                       575      - #           580      - #           585                  - - TCC AAA GCA AAG ATT CTG CTG GTT CTG TTT CA - #C CCC ATC AAC TCC TGT         1934                                                                       Ser Lys Ala Lys Ile Leu Leu Val Leu Phe Hi - #s Pro Ile Asn Ser Cys                   590          - #       595          - #       600                      - - GCC AAC CCC TTC CTC TAT GCC ATC TTT ACC AA - #A AAC TTT CGC AGA GAT         1982                                                                       Ala Asn Pro Phe Leu Tyr Ala Ile Phe Thr Ly - #s Asn Phe Arg Arg Asp               605              - #   610              - #   615                          - - TTC TTC ATT CTG CTG AGC AAG TGT GGC TGC TA - #T GAA ATG CAA GCC CAA         2030                                                                       Phe Phe Ile Leu Leu Ser Lys Cys Gly Cys Ty - #r Glu Met Gln Ala Gln           620                 6 - #25                 6 - #30                 6 -      #35                                                                              - - ATT TAT AGG ACA GAA ACT TCA TCC ACT GTC CA - #C AAC ACC CAT CCA        AGG     2078                                                                    Ile Tyr Arg Thr Glu Thr Ser Ser Thr Val Hi - #s Asn Thr His Pro Arg                          640  - #               645  - #               650              - - AAT GGC CAC TGC TCT TCA GCT CCC AGA GTC AC - #C AAT GGT TCC ACT TAC         2126                                                                       Asn Gly His Cys Ser Ser Ala Pro Arg Val Th - #r Asn Gly Ser Thr Tyr                       655      - #           660      - #           665                  - - ATA CTT GTC CCT CTA AGT CAT TTA GCC CAA AA - #C TAAAACACAA TGTGAAAAT    G   2179                                                                       Ile Leu Val Pro Leu Ser His Leu Ala Gln As - #n                                       670          - #       675                                             - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  695                                                              (B) TYPE: Amino acid                                                          (D) TOPOLOGY: Linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: signal se - #quence                                             (B) LOCATION: -17 to - #-1                                                    (C) IDENTIFICATION METHOD: - # hydrophobic                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: putative - #amino-terminal extracellular domain                 (B) LOCATION: 1 to 3 - #49                                                    (C) IDENTIFICATION METHOD: - # similarity with other                               dimeric g - #lycoprotein receptor extracellular                               domains, - #hydrophilic                                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: transmembran - #e domain                                        (B) LOCATION:   350 - #to 613                                                 (C) IDENTIFICATION METHOD: - # similarity to other G                               protein-coup - #led receptor transmembrane domains              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: putative - #transmembrane region I                              (B) LOCATION:   350 - #to 370                                                 (C) IDENTIFICATION METHOD: - # similarity to other G                               protein-coup - #led receptor transmembrane regions,                           hydrophobic, - # about 20-23 amino acids in length              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: putative - #transmembrane region II                             (B) LOCATION: 382 to - #404                                                   (C) IDENTIFICATION METHOD: - # similarity to other G                               protein-coup - #led receptor transmembrane regions,                           hydrophobic, - # about 20-23 amino acids in length              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: putative - #transmembrane region III                            (B) LOCATION: 427 to - #448                                                   (C) IDENTIFICATION METHOD: - # similarity to other G                               protein-coup - #led receptor transmembrane regions,                           hydrophobic, - # about 20-23 amino acids in length              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: putative - #transmembrane region IV                             (B) LOCATION: 469 to - #491                                                   (C) IDENTIFICATION METHOD: - # similarity to other G                               protein-coup - #led receptor transmembrane regions,                           hydrophobic, - # about 20-23 amino acids in length              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: putative - #transmembrane region V                              (B) LOCATION: 512 to - #533                                                   (C) IDENTIFICATION METHOD: - # similarity to other G                               protein-coup - #led receptor transmembrane regions,                           hydrophobic, - # about 20-23 amino acids in length              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: putative - #transmembrane region VI                             (B) LOCATION: 557 to - #580                                                   (C) IDENTIFICATION METHOD: - # similarity to other G                               protein-coup - #led receptor transmembrane regions,                           hydrophobic, - # about 20-23 amino acids in length              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: putative - #transmembrane region VII                            (B) LOCATION: 592 to - #613                                                   (C) IDENTIFICATION METHOD: - # similarity to other G                               protein-coup - #led receptor transmembrane regions,                           hydrophobic, - # about 20-23 amino acids in length              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: putative - #carboxy-terminal intracellular                           domain                                                                   (B) LOCATION: 614 to - #678                                          - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #2:                          - - Met Ala Leu Leu Leu Val Ser Leu Leu Ala Ph - #e Leu Ser Leu Gly Ser              -15          - #       -10          - #        -5                      - - Gly Cys His His Arg Ile Cys His Cys Ser As - #n Arg Val Phe Leu Cys           1             - #  5                - #   10               - #   15       - - Gln Glu Ser Lys Val Thr Glu Ile Pro Ser As - #p Leu Pro Arg Asn Ala                       20 - #                 25 - #                 30              - - Ile Glu Leu Arg Phe Val Leu Thr Lys Leu Ar - #g Val Ile Gln Lys Gly                   35     - #             40     - #             45                  - - Ala Phe Ser Gly Phe Gly Asp Leu Glu Lys Il - #e Glu Ile Ser Gln Asn               50         - #         55         - #         60                      - - Asp Val Leu Glu Val Ile Glu Ala Asp Val Ph - #e Ser Asn Leu Pro Lys           65             - #     70             - #     75                          - - Leu His Glu Ile Arg Ile Glu Lys Ala Asn As - #n Leu Leu Tyr Ile Asn      80                  - #85                  - # 90                 - # 95       - - Pro Glu Ala Phe Gln Asn Leu Pro Asn Leu Gl - #n Tyr Leu Leu Ile Ser                      100  - #               105  - #               110              - - Asn Thr Gly Ile Lys His Leu Pro Asp Val Hi - #s Lys Ile His Ser Leu                  115      - #           120      - #           125                  - - Gln Lys Val Leu Leu Asp Ile Gln Asp Asn Il - #e Asn Ile His Thr Ile              130          - #       135          - #       140                      - - Glu Arg Asn Ser Phe Val Gly Leu Ser Phe Gl - #u Ser Val Ile Leu Trp          145              - #   150              - #   155                          - - Leu Asn Lys Asn Gly Ile Gln Glu Ile His As - #n Cys Ala Phe Asn Gly      160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - Thr Gln Leu Asp Glu Leu Asn Leu Ser Asp As - #n Asn Asn Leu Glu        Glu                                                                                             180  - #               185  - #               190             - - Leu Pro Asn Asp Val Phe His Gly Ala Ser Gl - #y Pro Val Ile Leu Asp                  195      - #           200      - #           205                  - - Ile Ser Arg Thr Arg Ile His Ser Leu Pro Se - #r Tyr Gly Leu Glu Asn              210          - #       215          - #       220                      - - Leu Lys Lys Leu Arg Ala Arg Ser Thr Tyr As - #n Leu Lys Lys Leu Pro          225              - #   230              - #   235                          - - Thr Leu Glu Lys Leu Val Ala Leu Met Glu Al - #a Ser Leu Thr Tyr Pro      240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - Ser His Cys Cys Ala Phe Ala Asn Trp Arg Ar - #g Gln Ile Ser Glu        Leu                                                                                             260  - #               265  - #               270             - - His Pro Ile Cys Asn Lys Ser Ile Leu Arg Gl - #n Glu Val Asp Tyr Met                  275      - #           280      - #           285                  - - Thr Gln Thr Arg Gly Gln Arg Ser Ser Leu Al - #a Glu Asp Asn Glu Ser              290          - #       295          - #       300                      - - Ser Tyr Ser Arg Gly Phe Asp Met Thr Tyr Th - #r Glu Phe Asp Tyr Asp          305              - #   310              - #   315                          - - Leu Cys Asn Glu Val Val Asp Val Thr Cys Se - #r Pro Lys Pro Asp Ala                        - #  320               - #  325               - #  330                   - #  335                                                          - - Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr As - #n Ile Leu Arg Val Leu                      340  - #               345  - #               350              - - Ile Trp Phe Ile Ser Ile Leu Ala Ile Thr Gl - #y Asn Ile Ile Val Leu                  355      - #           360      - #           365                  - - Val Ile Leu Thr Thr Ser Gln Tyr Lys Leu Th - #r Val Pro Arg Phe Leu              370          - #       375          - #       380                      - - Met Cys Asn Leu Ala Phe Ala Asp Leu Cys Il - #e Gly Ile Tyr Leu Leu          385              - #   390              - #   395                          - - Leu Ile Ala Ser Val Asp Ile His Thr Lys Se - #r Gln Tyr His Asn Tyr      400                 4 - #05                 4 - #10                 4 -      #15                                                                              - - Ala Ile Asp Trp Gln Thr Gly Ala Gly Cys As - #p Ala Ala Gly Phe        Phe                                                                                             420  - #               425  - #               430             - - Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Th - #r Leu Thr Ala Ile Thr                  435      - #           440      - #           445                  - - Leu Glu Arg Trp His Thr Ile Thr His Ala Me - #t Gln Leu Asp Cys Lys              450          - #       455          - #       460                      - - Val Gln Leu Arg His Ala Ala Ser Val Met Va - #l Met Gly Trp Ile Phe          465              - #   470              - #   475                          - - Ala Phe Ala Ala Ala Leu Phe Pro Ile Phe Gl - #y Ile Ser Ser Tyr Met      480                 4 - #85                 4 - #90                 4 -      #95                                                                              - - Lys Val Ser Ile Cys Leu Pro Met Asp Ile As - #p Ser Pro Leu Ser        Gln                                                                                             500  - #               505  - #               510             - - Leu Tyr Val Met Ser Leu Leu Val Leu Asn Va - #l Leu Ala Phe Val Val                  515      - #           520      - #           525                  - - Ile Cys Gly Cys Tyr Ile His Ile Tyr Leu Th - #r Val Arg Asn Pro Asn              530          - #       535          - #       540                      - - Ile Val Ser Ser Ser Ser Asp Thr Arg Ile Al - #a Lys Arg Met Ala Met          545              - #   550              - #   555                          - - Leu Ile Phe Thr Asp Phe Leu Cys Met Ala Pr - #o Ile Ser Phe Phe Ala      560                 5 - #65                 5 - #70                 5 -      #75                                                                              - - Ile Ser Ala Ser Leu Lys Val Pro Leu Ile Th - #r Val Ser Lys Ala        Lys                                                                                             580  - #               585  - #               590             - - Ile Leu Leu Val Leu Phe His Pro Ile Asn Se - #r Cys Ala Asn Pro Phe                  595      - #           600      - #           605                  - - Leu Tyr Ala Ile Phe Thr Lys Asn Phe Arg Ar - #g Asp Phe Phe Ile Leu              610          - #       615          - #       620                      - - Leu Ser Lys Cys Gly Cys Tyr Glu Met Gln Al - #a Gln Ile Tyr Arg Thr          625              - #   630              - #   635                          - - Glu Thr Ser Ser Thr Val His Asn Thr His Pr - #o Arg Asn Gly His Cys      640                 6 - #45                 6 - #50                 6 -      #55                                                                              - - Ser Ser Ala Pro Arg Val Thr Asn Gly Ser Th - #r Tyr Ile Leu Val        Pro                                                                                             660  - #               665  - #               670             - - Leu Ser His Leu Ala Gln Asn                                                          675                                                              __________________________________________________________________________

We claim:
 1. Isolated DNA encoding a polypeptide which binds to humanFSH, comprising:(a) the nucleotide sequence shown in SEQ ID NO: 1; (b) amutant thereof which differs from (a) by substitution, deletion and/orinsertion of nucleotides which alter a total of no more than ten aminoacids in the polypeptide encoded by (a), which mutant encodes apolypeptide which binds human FSH; or (c) a fragment of (a) or (b)encoding a polypeptide which binds human FSH.
 2. A recombinantexpression vector comprising the DNA of claim
 1. 3. A cell transfectedwith the expression vector of claim
 2. 4. The cell of claim 3 which is amammalian cell.
 5. A method of producing human FSH receptor, or afragment or mutant thereof which binds FSH, which comprises culturingthe cell of claim 3 in a nutrient medium and optionally recovering thereceptor therefrom.
 6. The method of claim 5 wherein the cell is amammalian cell.
 7. The isolated DNA of claim 1, comprising thenucleotide sequence shown in SEQ ID NO:1 or a fragment thereof whichencodes a protein that binds human FSH.
 8. The isolated DNA of claim 7comprising the nucleotide sequence shown in Sequence ID No:1.
 9. Arecombinant expression vector comprising the DNA of claim
 8. 10. A celltransfected with the expression vector of claim
 9. 11. A method ofproducing human FSH receptor, or a fragment or mutant thereof whichbinds FSH, which comprises culturing the cell of claim 10 in a nutrientmedium and optionally recovering the receptor therefrom.
 12. Theisolated DNA of claim 7 comprising the nucleotide sequence shown inSequence ID NO:1 in which the nucleotide sequence encoding thecytoplasmic domain of the FSH receptor is deleted.
 13. A recombinantexpression vector comprising the DNA of claim
 12. 14. A cell transfectedwith the expression vector of claim
 13. 15. A method of producing ahuman FSH receptor portion which binds FSH, which comprises culturingthe cell of claim 14 in a nutrient medium under conditions that resultin the expression of said receptor portion and optionally recoveringsaid human FSH receptor portion.
 16. The isolated DNA of claim 7comprising the nucleotide sequence shown in Sequence ID NO:1 in whichthe nucleotide sequence encoding the cytoplasmic and transmembranedomains of the FSH receptor are deleted.
 17. A recombinant expressionvector comprising the DNA of claim
 16. 18. A cell transfected with theexpression vector of claim
 17. 19. A method of producing a human FSHreceptor portion which binds FSH, which comprises culturing the cell ofclaim 18 in a nutrient medium and recovering the receptor therefrom. 20.A recombinant expression vector comprising the DNA of claim
 12. 21. Acell transfected with the expression vector of claim
 20. 22. A method ofproducing a human FSH receptor which comprises culturing the cell ofclaim 21 in a nutrient medium and optionally recovering the receptortherefrom.
 23. An isolated DNA encoding a polypeptide which binds tohuman FSH, comprising a nucleotide sequence encoding:(a) the human FSHreceptor having SEQ ID NO: 2; (b) a polypeptide which includes theamino-terminal extracellular portion of the human FSH receptor, saidportion having the sequence of amino acids 1-349 of SEQ ID NO: 2; or (c)a polypeptide which includes a fragment of the human FSH receptor whichcomprises a substantial portion of said amino-terminal extracellularportion of the human FSH receptor and which retains substantially thesame FSH-binding characteristics as the complete extracellular portion.24. An isolated DNA in accordance with claim 23, comprising a nucleotidesequence encoding the human FSH receptor of SEQ ID NO:2.
 25. Arecombinant expression vector comprising the DNA of claim
 24. 26. A celltransfected with expression vector of claim
 25. 27. A method ofproducing a human FSH receptor portion which binds FSH, which comprisesculturing the cell of claim 26 in a nutrient medium under conditionsthat result in the expression of said receptor portion and optionallyrecovering said human FSH receptor portion.
 28. An isolated DNA inaccordance with claim 23, comprising a nucleotide sequence encoding apolypeptide of (b) or (c).
 29. A recombinant expression vectorcomprising the DNA of claim
 28. 30. A cell transfected with theexpression vector of claim
 29. 31. A method of producing a human FSHreceptor portion which binds FSH, which comprises culturing the cell ofclaim 30 in a nutrient medium under conditions that result in theexpression of said receptor portion and recovering said human FSHreceptor portion.
 32. A recombinant expression vector comprising the DNAof claim
 23. 33. A cell transfected with the expression vector of claim32.
 34. A method of producing a human FSH receptor or a portion thereofwhich binds FSH, which comprises culturing the cell of claim 33 in anutrient medium and optionally recovering the receptor therefrom.